The Potential of Spirulina platensis to Ameliorate the Adverse Effects of Highly Active Antiretroviral Therapy (HAART)

The human immunodeficiency virus (HIV) is one of the most prevalent diseases globally. It is estimated that 37.7 million people are infected with HIV globally, and 8.2 million persons are infected with the virus in South Africa. The highly active antiretroviral therapy (HAART) involves combining various types of antiretroviral drugs that are dependent on the infected person’s viral load. HAART helps regulate the viral load and prevents its associated symptoms from progressing into acquired immune deficiency syndrome (AIDS). Despite its success in prolonging HIV-infected patients’ lifespans, the use of HAART promotes metabolic syndrome (MetS) through an inflammatory pathway, excess production of reactive oxygen species (ROS), and mitochondrial dysfunction. Interestingly, Spirulina platensis (SP), a blue-green microalgae commonly used as a traditional food by Mexican and African people, has been demonstrated to mitigate MetS by regulating oxidative and inflammatory pathways. SP is also a potent antioxidant that has been shown to exhibit immunological, anticancer, anti-inflammatory, anti-aging, antidiabetic, antibacterial, and antiviral properties. This review is aimed at highlighting the biochemical mechanism of SP with a focus on studies linking SP to the inhibition of HIV, inflammation, and oxidative stress. Further, we propose SP as a potential supplement for HIV-infected persons on lifelong HAART.


Introduction
The human immunodeficiency virus (HIV) has continued to be a global public concern due to its widespread infection rate and alarming mortality rate [1]. The Joint United Nations Programme on HIV/AIDS (UNAIDS), in its most recent report in November 2021, estimated that 37.7 million people globally are living with HIV. It was also reported that about 1.5 million new HIV infections and 680,000 AIDS-related deaths have occurred in the year 2020 [1][2][3][4]. In South Africa, approximately 8.2 million people were living with HIV in the year 2021 [4]. According to the South African mid-year population statistics 2021, there has been an unprecedented increase from 79,420 to 85,154 HIV-AIDS-related deaths in 2021 [4]. Recently, the easy availability of antiretrovirals (ARVs) has tremendously changed the pattern of death. ARVs have also helped prolong the lifespan of HIV-infected people in South Africa. Globally, about 27.5 million HIV-infected persons had access to ARVs in 2020, while approximately 5.6 million infected South Africans accessed ARVs in 2020 [1, 4,5].
The highly active antiretroviral therapy (HAART) entails combining three or more antiretroviral drugs that are subject to the HIV-infected person's viral load. HAART assists in regulating viral loads and preventing the progression to AIDS. Despite its success in prolonging HIV-infected patients' lifespans, the use of HAART promotes metabolic syndrome (MetS) through an inflammatory pathway, excess production of reactive oxygen species (ROS), and mitochondrial dysfunction. Over three decades since its discovery, HAART has scription, chemokine activation, and the survival of cells [7]. NF-κB is also a well-known mediator of inflammation that promotes HIV transcription [7,46].
Inflammation linked to HAART is evidenced by the persistently high levels of interleukin 6 (IL-6), C-reactive proteins, and D-dimers [47]. HIV creates chronic pro-inflammatory conditions that promote MetS [7]. Studies have shown that HIV is associated with inflammation, apoptosis, and mitochondrial dysfunction [7,48]; however, the mechanism that links HIV with MetS remains unclear [7]. Herein, we highlight the significance of SP as an anti-inflammatory supplement for HIV-infected people on lifelong HAART and its mechanism of inhibition on MetS.

Spirulina Species
Spirulina has three commonly investigated species due to their potential therapeutic nature and high nutritional content. These Spirulina species include Spirulina platensis (SP) (otherwise known as Arthrospira platensis), Spirulina maxima (Arthrospira maxima), and Spirulina fusiformis (Arthrospira fusiformis). These Spirulina species are also classified as oxygenic photosynthetic bacteria under Cyanobacteria and Prochlorales [49][50][51][52][53][54]. SP is found in alkaline water with abundant bicarbonate and saline [22,55]. Spirulina species are generally three-dimensional helix microstructures [56] protected by a cell wall composed of complex sugars and proteins [22]; however, helical transformation results after mature trichomes divide into hormogonia, binary fission, and undergo length elongation [57]. SP is considered an antioxidant and anti-inflammatory agent [58]. It is also considered as a nutraceutical food supplement due to its high content of proteins, vitamins, and minerals. Moreover, its composition includes chlorophyll, phycocyanin, and carotenoid. Chlorophyll has antioxidant and antimutagenic properties [59,60], carotenoids are vitally important antioxidants with cancer-inhibiting abilities [53], and phycocyanin is a Bili protein with antioxidant and radical scavenging properties [61]. Moreso, SP has also been credited for its cancer-and viral infection-suppressing abilities [62].

Role of Spirulina in the Inhibition of Oxidative Stress
Recently, research has unveiled the important roles of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the production of reactive oxygen species (ROS). NADPH is contained in the nervous system and its assemblage and activation generate free radicals (FR) which subsequently destroy cells. Spirulina is a potent inhibitor of NADPH oxidase, which has been one of the proteins responsible for the production of ROS and subsequent oxidative stress [54,63,64]. SP has the potential to inhibit oxidative stress by blocking NADPH oxidase [21,25,28,29] and to enhance (Figure 1) mitochondrial health by promoting an antioxidant response [30][31][32][33]. Furthermore, SP prevents FR-induced apoptotic cell death through the inhibition of oxidative stress [65].

Multitargeted Therapeutic Roles of SP
SP has a therapeutic effect against vascular diseases, cancer, diabetes, neurodegenerative diseases, and inflammatory disorders [66]. Spirulina treatment enhances the NF-kappa B-directed luciferase expression [67]. It has antiallergic effects [54], including against allergic rhinitis in humans [68]. Spirulina is an immune booster [22]. It prevents cellular aging, infectious diseases, and promotes a strong immune system [57]. Spirulina has central neuroprotective effects in rodents [69]. It is also associated with the inhibitory effects against numerous viruses, such as HIV-1, herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), influenza type A, measles, and other enveloped viruses [53,[70][71][72][73]. Moreover, it has antimutagenic and anticancer effects [57]. Spirulina is an effective treatment against chronic arsenic poisoning with melanosis and keratosis [74]. SP shares similar chemical structures and physiological activities with bilirubin [25][26][27][28]75]. SP antioxidant properties are due to its composition and the presence of phycobilins, phycocyanin, and phycocyanobilin [25-27] ( Figure 2). Phycobilins are similar in structure to bile pigments such as bilirubin, a well-known ROS scavenger [22,76]. Phycocyanin has been proven to possess antioxidant and anti-inflammatory activities [34,54,63,64,77,78]. Phycocyanin is also structurally similar to biliverdin, a strong inhibitor of NADPH oxidase and inflammation-induced radicals [25,34]. A study conducted by Zheng et al. (2013) indicated that phycocyanin normalized urinary and renal oxidative stress markers and the expression of NADPH oxidase components. Furthermore, phycocyanobilin, bilirubin, and biliverdin inhibited NADPH-dependent superoxide production in renal mesangial cells [25]. The study also demonstrated that SP may be used in a therapeutic approach to prevent diabetic nephropathy through the inhibition of oxidative stress [25]. Thus far, phycocyanine, the most powerful natural antioxidant, is only present in cyanobacteria and thus in spirulina.  SP exhibited neuroprotective activities through antioxidant and anti-inflammatory effects [79]. It has also shown significant antioxidant activity in vitro by scavenging nitric oxide and preventing DNA damage by scavenging hydroxyl radical ( Figure 3) [80]. Antidiabetic and anti-inflammatory properties of SP [81] are based on its significant free-radical scavenging activities. SP contains compounds that fall under a broad spectrum of antioxidant agents, such as alkaloids, flavonoids [82], and phycocyanin [28, [83][84][85][86]. Moreso, it provides trace minerals for the synthesis of antioxidant enzymes, demonstrated by the antidiabetic response in rats [87]. It has potential benefits in assisting with the reduction in chronic inflammatory conditions [88]. Spirulina incorporated into skin cream showed promising results as an anti-inflammatory and a wound-healing agent [89]. Spirulina against HIV-1 demonstrated its ability as an antiviral compound. Studies have demonstrated the ability of SP in the inhibition of HIV-1 replication in human T-cell lines, peripheral blood mononuclear cells (PBMC), and Langerhans cells (LC). The inhibition of the viral production by spirulina extract (between 0.3 and 1.2 µg/mL) was found to be approximately 50% in PBMCs [57]. More studies are needed to fully understand the mechanism behind the inhibition of HIV by SP.
SP promotes the activation and expression of heme oxygenase 1 (HO-1) and endothelial nitric oxide synthase (eNOS) [104,105]. HO-1 is suggested to play an important part in the adaptive reprogramming which could result in Nrf-2 activation, but this pathway is unclear, and more studies are required. Moreso, SP causes the activation of the Nrf2/HO-1 pathway [106]. Nrf-2 activation by SP results in the production and increased expression of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) [100].

Mechanism of HAART-Induced Oxidative Stress, Inflammation, and Mitochondrial Dysfunction
The exact mechanism of HAART-induced oxidative stress has not been completely explored; however, studies have demonstrated the link between HAART use and oxidative stress. HAART is linked with lipid metabolism dysfunction through the induction of peripheral lipodystrophy. Lipodystrophy results from the impaired cytoplasmic retinoic-acid binding protein type 1 (CRABP1)-mediated cis-9-retinoic acid stimulation of peroxisome proliferator-activated receptor type gamma (PPAR-γ), leading to impaired differentiation and increased apoptosis of peripheral adipocytes. HIV-1 protease-inhibitors further inhibit the cytochrome P450 3A-mediated synthesis of cis-9-retinoic acid, one of the key activators of PPAR-γ [166]. Insulin resistance occurs following impaired fat storage and lipid release [141,142,166], which impacts the oxidant profile. The depletion of ATP production and mitochondrial dysfunction [8,9], and the depletion of mitochondrial DNA [167][168][169], are some of the ways HAART induces oxidative stress. HIV increases oxidative stress, and HAART increases lipid oxidation, which amplifies the ROS imbalance leading to increased oxidative stress complications [10,170].
Chronic exposure to HAART induces increased oxidative stress in endothelial cells and mononuclear cell recruitment, which leads to cardiovascular diseases in HIV patients on ARV therapy [12]. Inducing oxidative stress is common for protease and reverse transcriptase inhibitors [171]. HAART drugs induce oxidative stress in various ways, which include inhibiting DNA pol-γ activity and leading to mitochondrial dysfunction, and also through the depletion of mitochondrial DNA [167][168][169]. Studies have shown that patients on HAART have abnormally high levels of free oxygen radicals in sera compared to untreated HIV patients and HIV-uninfected participants [10,11,168,170,172,173].
Adverse drug reactions vary; TDF and lopinavir cause acute and chronic renal dysfunction [174][175][176][177][178]. TDF inhibits mitochondrial DNA-polymerase gamma, hence leading to the impaired function of energy-dependent transporters [179,180]. NRTIs are associated with the inhibition of mitochondrial DNA polymerase, lactic acidosis, subcutaneous lipoatrophy, peripheral neuropathy, and pancreatitis. The level of mitochondrial toxicity depends on the drugs; it is low with 3TC, FTC, and TDF, followed by ZDV, and higher with d4T. NNRTIs are associated with life-threatening skin reactions and toxic hepatitis. PIs are associated with insulin resistance and hyperlipidemia [7,114].

Combined and Synergistic Therapeutic Actions of HAART and SP
Studies have shown that the possible therapeutic effects of antioxidants may provide strategies in suppressing oxidative stress-induced comorbidities that emerge with the use of HAART therapy in HIV-infected individuals [12]. The combination of HIV and HAART has been associated with increased oxidative stress and lipid peroxidation. Furthermore, HIV or HAART induces ROS by inducing NADPH oxidase [181,182]. Interestingly, SP is a potent antioxidant [26,27] with anti-inflammatory activities [34], which makes it a potential supplement in the mitigation of oxidative stress induced by HAART adverse drug reactions. Moreover, SP can inhibit NADPH oxidase which is considered one of the main sources of ROS and free radicals in HIV-infected persons on HAART [34, 181,182], resulting in reduced oxidative stress [28]. Moreover, β-carotene from SP protects against singlet oxygen-mediated lipid peroxidation [101]. Among HAART complications, TDF and lopinavir cause acute and chronic renal dysfunction [174][175][176][177][178]. Herein, phycocyanin from SP can normalize urinary and renal oxidative stress markers and inhibit NADPH-dependent superoxide production in renal mesangial cells [25], ameliorating renal dysfunction. Lately, SP has been an effective therapeutic approach to preventing diabetic nephropathy through the inhibition of oxidative stress [25]. These properties indicate SP as a potential agent to mitigate renal dysfunction caused by HAART. As mentioned above, NRTIs can inhibit mitochondrial DNA polymerase [179,180]. Studies in vitro showed that SP can enhance cell nucleus enzyme function, repair DNA synthesis [57], and enhance mitochondrial health [30][31][32][33]80]. Mitochondrial toxicity presented as peripheral neuropathy and lactic acidosis can be corrected by SP through providing trace minerals for the synthesis of antioxidant enzymes [87] and reducing chronic inflammatory conditions [88].
NNRTIs are associated with life-threatening skin reactions and toxic hepatitis [114], these conditions may be ameliorated by SP. Phycocyanin from SP can inhibit liver microsomal lipid peroxidation [28, [83][84][85][86][90][91][92][93][94][95][96][97], and hence reducing toxic hepatitis. Moreso, SP incorporated into skin creams showed promising results as an anti-inflammatory and a wound-healing agent [89]; this can be beneficial in the mitigation of NNRTI-induced skin reactions. PI therapy induces insulin resistance and hyperlipidemia [7]. Additionally, HAART may be associated with a higher risk of myocardial infarction [114,183,184]. SP has a therapeutic effect against vascular diseases, cancer, diabetes, and neurodegenerative diseases [66]. In addition, the Spirulina family has also shown central neuroprotective effects in rodents [69] and may exert its neuroprotective activities through antioxidant and anti-inflammatory effects [79]. Therefore, SP is a recommended antioxidant to use as a supplement; the list of benefits is evident. It also has antiallergic effects [54], prevents cellular aging and infectious diseases, and promotes a strong immune system [57]. Herein, promotion of a strong immune system by SP can help increase CD4 cell counts, lower HIV viral loads, and slow down the progression to AIDS. Moreover, SP prevents FR-induced apoptotic cell death [65]; this may help decrease apoptosis of peripheral adipocytes induced by HAART. Chemically, SP is a recommended source of proteins, vitamins, and minerals [57], important nutrients for individuals on the HAART program. Finally, SP can assist HAART in the inhibition of HIV-1 replication because it has been shown to inhibit viral production in PBMCs.
There has been a number of clinical studies to investigate whether SP improves the quality of life in HIV-infected individuals. Marcel (2011) reported that insulin sensitivity in HIV patients improved more when a spirulina nutritional supplement was used instead of soybean [185]. Another study demonstrated for the first time that spirulina improves antioxidant capacity in people living with HIV [186]. Spirulina supplementation combined with a qualitative balanced diet showed potential to inhibit lipid abnormalities [187], significantly increase CD4 cells, and reduce the viral load in HIV-infected antiretroviralnaive patients [187][188][189]. However, there is limited information on the investigation of SP confirming the mechanism of antioxidant and anti-inflammatory effects and the impact on the quality of life in the HIV-positive population taking HAART. Thus far, it has been shown that supplementation with Spirulina platensis could improve the immune status of HIV patients on ART and decrease inflammatory and pro-oxidant levels [190]. The development of more clinical studies to confirm the SP protective effect in this population will answer many questions. The recommended concentrations of SP for daily supplementation varies, as studies have successfully used 19 g [185], 5 g [186], and 10 g [190,191].

Conclusions
HIV continues to be a major global cause of mortality. Besides the therapeutic benefits of HAART in HIV treatment, HAART has been linked to numerous adverse drug reactions which include oxidative stress, inflammation, and the disruption of mitochondrial function. SP as an antioxidant, anti-inflammatory, anticancer, and nutritional supplement possesses various corrective properties against attacks from viruses and bacteria. The corrective health properties of SP are largely attributed to antioxidant pigments found in SP. These pigments include chlorophyll, carotenoids, and phycocyanin which facilitate antioxidant, anti-inflammatory, and anticancer properties. The corrective properties of SP indicated in this review highlight its potential in the mitigation of HAART adverse drug reactions and MetS. The SP supplement potential is also supported by its ability to assist HAART in the inhibition of HIV-1. This review highlighted the corrective properties of potent antioxidant SP potential as a supplement for individuals on lifelong HAART experiencing MetS. Furthermore, this review highlights the need for more studies on SP and HAART synergy.